Mechanisms of protein sorting are of fundamental importance in differentiation, and mammalian erythropoiesis provides a valuable model to study this phenomenon. The erythroid proteins spectrin and band 3, for example, remain with the mature reticulocyte, while most surface proteins segregate with the extruded nucleus. The molecular basis for these differences is obscure. Spectrin is present throughout development, whereas band 3 is induced 2-3 days before enucleation. These developmental changes lead to the maturation of the cell membrane and cytoskeleton. The two- dimensional network of spectrin is reinforced by band 4.1 and short chains of actin. Spectrin is also linked to band 3 via ankyrin. This arrangement restricts the lateral mobility of the integral membrane protein. It is likely that the orderly assortment of these proteins noted with red cell development is influenced critically by their mutual interactions. This proposal will address the following: a) Does gelsolin, an actin-serving protein, regulate the length of F-actin in mammalian red cells, and does perturbation of actin polymerization interfere with protein sorting or cytoskeletal stability? b) Is the segregation of band 3 with the mature red cell a consequence of its binding to the cytoskeleton, and does it play a role in the long-term stability of the cytoskeleton? Specifically, a survey of the following proteins will be made to assess their assortment during development in MEL and mouse bone marrow cells: actin, spectrin, band 4.1, ankyrin, band 3, and gelsolin. Next, to modulate actin polymerization in vivo, MEL cells will be transfected with appropriate gelsolin construct to augment or suppress actin- severing activity at different stages of differentiation. Analogous studies with band 3 will also be performed to modulate its expression at various stages of erythropoiesis. The effects of these manipulations on cell morphology, osmotic fragility, enucleation, and protein assortment will be evaluated. In addition to probing basic mechanisms of protein sorting, these studies should improve our understanding of hemolytic anemias by providing cell culture models for these disorders.